This invention relates to fluorescence imaging and, more particularly, to selective fluorescence imaging using fluorescence spectral properties preselected by flow cytometer analysis.
Fluorochromes are frequently bound to molecular cell components to yield quantitative information about the cell components. Several different fluorochromes may be present in a given cell sample to yield a number of emission spectra. Further, the emission spectrum from a given fluorochrome may be altered as a result of changes in the physiological state of the cells.
Fluorescence is generally detected by one or more detectors that are responsive to specific wavelengths of interest. Cytometry, either flow cytometry or image cytometry, may use this fluorescence as some measure of individual cells or cellular constituents. Cytometry can differentiate between effects that affect all cells equally and effects that cause subpopulations of cells within the population to change. See generally Clinical Cytometry, M. Andreeff ed., New York Academy of Sciences, New York, N. Y. (1986), incorporated herein by reference. It will be appreciated that an increasing number of fluorochromes with different sensitivities to changes in cell physiology and labeled monoclonal antibodies that recognize specific antigens impose a requirement for an increasing number of spectral channels for fluorescence analysis.
U.S. Pat. No. 4,905,169, issued Feb. 27, 1990, to Buican et al., incorporated herein by reference, teaches cytometry apparatus, and particularly flow cytometry apparatus, for simultaneously resolving a plurality of spectral properties from a total fluorescence spectrum. A Fourier-Transform (FT) spectrometer encodes spectral information in a waveform (interferogram) on which it then performs a numerical, rather than optical, spectral analysis. When installed in a flow cytometer, the FT spectrometer analyzes the total fluorescence emitted by single particles as they cross an excitation laser beam and resolves this emission into a set of separate fluorescences, each described by a characteristic emission spectrum which has been selected by the operator. Thus, a set of numbers (spectral parameters) is produced for each particle, describing the spectral properties of the individual particles in the sample. Taken separately, each spectral parameter represents an enhancement of a specific fluorescence, since it is obtained by eliminating all other emission spectra from the total fluorescence.
Image cytometry devices are also used for detecting fluorochromes in biological specimens and may be implemented as a laser-scanning confocal microscope. See generally Handbook of Biological Confocal Microscopy, J. Pawley ed., Plenum Press, New York, N.Y. (1989, 1990), incorporated herein by reference. However, fluorescence data is obtained on a pixel-by-pixel basis over the biological sample and it is difficult to quantify specific cell subpopulations in the sample. This is normally done by physically sorting a selected subpopulation by flow sorting, recovering the cells, and placing them on a slide for morphological analyses. Accordingly, the present invention incorporates Fourier-Transform technology into the laser-scanning confocal microscope to enable image enhancement for a selected superposition of fluorochrome spectra to effect a virtual sorting of a subpopulation previously identified.
It is an object of the present invention to generate concurrent multiple images from a laser-scanning confocal microscope, each enhanced for a particular emission spectrum.
It is another object of the present invention to generate an image from a biological specimen enhanced for an emission spectrum selected from flow cytometer data.
One other object of the present invention is to enable a virtual sorting of cells in a biological sample without physical sorting.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.